Reducible compounds which provide leuco dyes for analytical compositions, elements and methods of using same

ABSTRACT

Certain reducible compounds are useful in analytical compositions, elements and methods, for example for the detection of bacterial cells. These compounds comprise a moiety which provides a leuco dye upon reduction. This lecuo dye can then be oxidized with additional molecules of reducible compound in order to provide a detectable dye. Thus, these reducible compounds are considered bioamplifiers from which a multiplicity of dye molecules can be provided from a single reducible compound molecule. Structurally, the reducible compounds are quinones having suitable substituents which promote varying amounts of leuco dye release at physiological pH.

FIELD OF THE INVENTION

This invention relates to clinical chemistry. In particular, it relatesto novel reducible compounds and to their use in dry or wet assays ofliquids, such as biological fluids, to release useful leuco dyes whichcan be oxidized to provide dyes. These compounds are especially usefulin the detection of microorganisms.

BACKGROUND OF THE INVENTION

Chemical analysis of liquids, such as water, milk and biological fluidsis often desirable or necessary for health maintenance and diagnosticcare. Various compositions and elements to facilitate such analyses areknown. Such compositions and elements generally include a reagentcomposition for determining a substance under analysis, identified as an"analyte" herein. The analyte can be a living organism or a nonlivingchemical substance. The reagent composition, upon interaction with theanalyte, provides a detectable change (for example, dye formation).

Recently, much work has been directed to developing compositions andelements which are useful for rapid and highly quantitative diagnosticor clinical analysis of biological fluids such as whole blood, serum,plasma, urine and the like.

For example, for the rapid and effective diagnosis and treatment ofinfectious diseases, it is desirable to be able to detect the bacteriacausing the disease as rapidly as possible. Infections of the urinarytract are among the most common bacterial diseases, second in frequencyonly to infections of the respiratory tract. In fact, in many hospitals,urinary tract infections are the most common form of nosocomialinfections, often following the use of in-dwelling catheters and varioussurgical procedures. Most urinary tract infections result from ascendinginfection by microorganisms introduced through the urethra and vary inseverity from an unsuspected infection to a condition of severe systemicdisease. Such infections are usually associated with bacterial counts of100,000 (10⁵) or more organisms per ml of urine, a condition referred toas significant bacteriuria. Under normal conditions, urine is sterile,although contamination from the external genitalia may contribute up to1,000 (10³) organisms per ml in properly collected and transportedspecimens.

Significant bacteriuria may be present in a number of pathologicalconditions involving microbial invasion of any of the tissues of theurinary tract, or may result from simple bacterial multiplication in theurine without tissue invasion. The infection may involve a single sitesuch as the urethra, prostrate, bladder, or kidney, although frequentlyit involves more than one site. Infection restricted to the urine maypresent itself as asymptomatic bacteriuria, that is, a condition whichmanifests no overt signs or symptoms of infection. Early treatment ofthis condition can prevent the development of more serious conditions,for example pyelonephritis (inflammation of the kidney and the renalpelvis). The rapid detection of bacteria by a reliable method wouldtherefore facilitate an early and specific diagnosis.

Further, in order to insure that a prescribed antibiotic is in facteffective in treating an infection, repeated tests during therapy arerequired. The need for simple, rapid bacteriuria tests is thus clear.Moreover, in view of the frequent unsuspected asymptomatic occurrencesof urinary tract infections among children, pregnant women, diabeticsand geriatric populations, diagnosis of which may require collection andtesting of several specimens, bacteriuria tests must be sufficientlysimple and economical to permit routine performance. Again, thisillustrates the need for a rapid and inexpensive bacteriuria detectionmethod.

Current laboratory methods based on culturing microorganisms, forexample, the calibrated loop-direct streak method, require significantincubation periods (18-24 hours) before results can be determined. Theselaboratory methods are also time-consuming to perform and requireconsiderable clinical training and facilities.

Known commercial methods for relatively rapid detection of bacteriuriahave serious drawbacks. They are tedious, not completely reliable,require complex reagents or instrumentation, and have limitedsensitivity to certain microorganisms and susceptibility to drug orother interferences. Hence, the usefulness of known methods is severelylimited.

It is also known that colorless materials, for example, tetrazoliumsalts, can be reduced by micororganisms to form a colored formazan dye,as described in U.S. Pat. No. 3,415,718 (issued Dec. 10, 1968 to Forkmanet al). However, the use of formazan dyes for detecting microorganismshas several drawbacks. The formazan dyes generally have low extinctioncoefficients and therefore cannot be used to detect low levels ofmicroorganisms. The tetrazolium salts have structures that are notreadily modified to increase the extinction coefficients of the formazandyes. Some formazan dyes are insoluble in water and can be toxic to themicroorganisms.

U.S. Pat. No. 4,144,306 (issued Mar. 13, 1979 to Figueras) describes amultilayer element for analysis of liquids. This element can include aninteractive composition which interacts with an analyte to release apreformed, detectable moiety from an immobile carrier nucleus uponoxidation or reduction. Such release generally requires the presence ofa highly alkaline medium (i.e. pH greater than 13). The spectralabsorption band of the preformed detectable moiety is the same beforeand after release, and radiation-blocking layers are used in the elementto screen out unwanted absorption from unreleased detectable moietyduring the assay.

Copending and commonly assigned U.S. Ser. No. 824,766, filed Jan. 31,1986 by Belly et al describes reducible compounds which when reducedrelease detectable moieties, such as dyes. These reducible compounds canbe used to detect microorganisms or other analytes which will reduce thecompounds at physiological pH (generally a pH from 4 to 9). Whileproviding an advance in the art for detecting such materials, thecompounds of Belly et al require one molecule of reducible compound foreach molecule of detectable species released. This feature may besufficient for many analytes which are found in samples in relativelyhigh concentrations. However, where the analyte is present at lowconcentrations, or where an enhanced signal is required, it would bedesirable to have reducible compounds which provide increased signal orcan be amplifiable in some manner.

SUMMARY OF THE INVENTION

The problems noted above are overcome using a novel reducible compoundof the structure CAR--R¹ wherein CAR-- is the quinone structure ##STR1##R² and R⁴ are independently hydrogen, alkyl, aryl or an electronwithdrawing group,

R⁵ is methylene,

R³ is the same as --R⁵ --R¹, or is hydrogen, alkyl, aryl or an electronwithdrawing group, or R³ and R⁴, taken together, represent the atomsnecessary to complete a fused carbocyclic ring, and

R⁶ is aryl, and

R⁷ is substituted aryl, anilino, naphthylimino or an activated methylenegroup, or R⁶ and R⁷, taken together with the nitrogen atom to which theyare attached, represent the carbon and hetero atoms which, after releaseof R¹ from the quinone nucleus and decarboxylation, form a leuco dye.

The reducible compound noted above can be used in a composition which isbuffered at a pH of 9 or less with one or more appropriate buffers.Alternatively, the compound can be incorporated in a dry analyticalelement for the determination of an analyte. This element comprises anabsorbent carrier material, and one or more reagent zones in which thereducible compound is located.

A method for the determination of an analyte comprises the steps of:

A. at a pH of 9 or less, contacting a sample of a liquid suspected ofcontaining an analyte with the reducible compound noted above, toprovide a leuco dye,

B. oxidizing the provided leuco dye with additional molecules of thereducible compound to provide a dye, and

C. detecting the dye provided as a result of the presence of the analytewhich effects the release of R¹ from the reducible compound.

The novel reducible compounds of this invention provide the advantage ofproducing many molecules of dye for each molecule of analyte (that is,for each electron received from an analyte). Such compounds, then can beused to advantage in analytical procedures where the analyte is presentin relatively low concentrations, or where amplification is needed toproduce a large signal with small amounts of reagents. The compounds ofthis invention provide these advantages because of the unique R¹ moietywhich contains the oxycarbonyl linkage between the quinone nucleus andR⁶ and R⁷ attached to the nitrogen atom. The leuco dye released when thereducible compound is reduced is then oxidized by unreduced reduciblecompound to provide a desired dye. This cycle, begun with a singleelectron transfer, of leuco dye formation and subsequent dye formationcan be continued as long as there is reducible compound present.

DETAILED DESCRIPTION OF THE INVENTION

The reducible compounds of this invention are broadly defined ascompounds which can be reduced, preferably at a physiological pH (suchas from 6 to 9) to release a moiety which becomes a leuco dye (that is,a dye precursor). A molecule of this leuco dye is readily oxidized toprovide a dye while acting to reduce a second molecule of reduciblecompound which begins the cycle again. The net result is a multiplicityof dye molecules from a single electron from an analyte or its reactionproducts.

More particularly, the compounds of this invention are defined by thestructure CAR--R¹ wherein CAR-- is the quinone structure ##STR2##

In the quinone nucleus illustrated above, R² and R⁴ are independently(that is, the same or different) hydrogen, substituted or unsubstitutedalkyl of 1 to 40 carbon atoms (for example methyl, ethyl, hydroxymethyl,methoxymethyl, benzyl and others apparent to one skilled in the art)substituted or unsubstituted aryl (for example phenyl, naphthyl,methylnaphthyl, p-nitrophenyl, m-methoxyphenyl, phenylsulfonamido andothers apparent to one skilled in the art) or an electron withdrawinggroup which generally has a positive Hammett sigma value, and preferablyhas a sigma value greater than about 0.06. Hammett sigma values arecalculated in accordance with standard procedures described, for examplein Steric Effects in Organic Chemistry, John Wiley & Sons, Inc., 1956,pp. 570-574 and Progress in Physical Organic Chemistry, Vol. 2,Interscience Publishers, 1964, pp. 333-339. Representative electronwithdrawing groups having positive Hammett sigma values include cyano,carboxy, nitro, halo (fluoro, bromo, chloro or iodo), trihalomethyl (forexample trifluoromethyl, or trichloromethyl), trialkylammonium,carbonyl, carbamoyl, sulfonyl, sulfamoyl, esters and others readilyapparent to one skilled in the art, or alkyl or aryl groups (definedabove) substituted with one or more of these electron withdrawinggroups. Preferred electron withdrawing groups include p-nitrophenyl,m-nitrophenyl, p-cyanophenyl and 2,5-dichlorophenyl. Aryl groups withmethoxy or acetamido groups in the meta position are also useful.

R³ is the same as --R⁵ --R¹, or it is hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted aryl or an electronwithdrawing group as defined above for R² and R⁴. Alternatively, R³ andR⁴, taken together, represent the atoms necessary to complete asubstituted or unsubstituted fused carbocyclic ring attached to thequinone nucleus. For example, such a ring (mono- or bicyclic) can havefrom 4 to 8 carbon atoms in the backbone.

R⁵ is methylene which can be unsubstituted or substituted with methyl ormethoxy.

R⁶ is a substituted or unsubstituted aryl of 6 to 10 carbon atoms, suchas phenyl, 4-aminophenyl, naphthyl, p-dimethylaminophenyl,p-hydroxyphenyl or 4-dimethylaminonaphthyl and others known to oneskilled in the art.

R⁷ can be aryl of 6 to 14 carbon atoms in the nucleus which issubstituted with substituents which provide with the nitrogen atom aleuco dye moiety (such as hydroxy and tertiary amino), or it can besubstituted or unsubstituted anilino, substituted or unsubstitutednaphthylimino (such as anilino, p-nitroanilino, naphthylimino or4-sulfonylmethylnaphthylimino), or an activated methylene group (such asα-benzoylacetanilide)

Alternatively, R⁶ and R⁷ taken together with the nitrogen atom to whichthey are attached, represent the carbon and hetero (nitrogen, oxygen,selenium, sulfur, phosphorus) atoms which, after release of R¹ from thequinone nucleus and decarboxylation, form a leuco dye. Such leuco dyemoieties (that is, R⁶, R⁷ with the nitrogen atom) are reduced forms ofdyes, such as leuco azo dyes, leuco azomethine dyes, leuco azoanilinesor diarylamine compounds.

Representative examples of useful --NR⁶ R⁷ groups include, but are notlimited to, the following: ##STR3## wherein R⁸, R⁹, R¹⁰ and R¹¹ areindependently of each other any of the substituents such as nitro,alkylsulfonyl (of 1 to 3 carbon atoms), tertiary amino, alkoxy (of 1 to3 carbon atoms), alkyl (of 1 to 3 carbon atoms, and others known to oneskilled in the art), and R¹² is hydroxy or tertiary amino.

When the reducible compound of this invention is reduced, the R¹ moietyis released. Since this moiety contains an unstable carbamic acidlinkage, it spontaneously undergoes decarboxylation to form a leuco dye.

The preferred reducible compounds of this invention have at least twoelectron withdrawing groups for R², R³ and R⁴ as defined above, or R³and R⁴, taken together, represent the atoms necessary to complete asubstituted or unsubstituted fused 5- to 7-member carbocyclic ring asdefined above. More preferably, R⁵ is unsubstituted methylene and R³ andR⁴ are the 5- to 7-membered carbocyclic ring as defined above.

Representative reducible compounds of this invention include, but arenot limited to: ##STR4##

Compound I is preferred.

The novel reducible compounds of the present invention are preparedusing a sequence of individually known reactions. Generally, thepreparation sequence includes the following general steps: (1)preparation of the eventual dye, (2) hydrogenation of the dye to a leucodye, (3) phosgenation of the leuco dye and (4) reaction of the leuco dyecarbamyl chloride and a hydroxymethyl CAR-- derivative. Representationpreparations are provided in Examples 1 and 2 below.

Generally, the reducible compounds described herein have limited watersolubility. Hence, it is best, when using them in an aqueousenvironment, to prepare a dispersion of the compound prior to use. Suchdispersions generally comprise the reducible compound, an aqueous buffersolution and either a water-solubilizing surfactant or a water-miscibleorganic solvent for the compound, or both.

Surfactants which are useful in the practice of this invention includeany surfactants which do not inhibit compound reduction. Generally, fordetection of living cells, the useful surfactants are nonionicsurfactants, including, for example, alkylarylpolyethoxy alcohols (forexample, TRITON X-100 and X-305 available from Rohm & Haas),p-alkylaryloxypolyglycidols (for example, SURFACTANT 10G available fromOlin Corp.), TWEEN 80 (available from ICI Americas, Inc.), and othersknown to one skilled in the art.

Useful water-miscible organic solvents include, but are not limited to,alcohols (for example, methanol, ethanol, propanol and others known inthe art), N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile orhexamethylenephosphoramide. The particular solvent to be used for aparticular reducible compound can be readily determined by routineexperimentation.

A dispersion can be prepared in the following general manner with theparticular details of such a preparation illustrated in Example 3 below.The reducible compound is dissolved in the water-miscible solvent at aconcentration which depends upon its molecular weight, but generally atfrom about 1 to about 100, and preferably from about 5 to about 80, mgper ml of solvent. The resulting solution is then mixed with a suitablesurfactant in an amount generally of from about 0.1 to about 24, andpreferably from about 0.5 to about 10, mg surfactant per ml ofdispersion. This preparation is generally carried out at roomtemperature.

These dispersions generally contain a buffer in an amount effective topreferably maintain a physiological pH (such as 6 to 9). Theconcentration of buffer in the dispersion can vary widely, but isgenerally from about 0.01 to about 0.1 molar. Representative buffersinclude phosphates, borates and others reported by Good et al inBiochemistry, 5, 467 (1966), and Anal. Biochem., 104, 300 (1980).

The reducible compounds described herein are useful in compositions foranalytical determination (that is, qualitative or quantitativedetection) of aqueous and nonaqueous liquids, for example, biologicalfluids, manufacturing processes, wastewater or food stuffs.Determinations can be made of various analytes using a single reactionor a sequence of reactions which bring about reduction of the compoundand provision of the leuco dye. The various analytes include livingcells (for example, bacteria, white blood cells, yeast or fungi),enzymes (for example, lipase, glucose oxidase, lactate oxidase, creatinekinase, α-glycerophosphate oxidase, lactate dehydrogenase, pyruvatedehydrogenase, glucose-6-phosphate dehydrogenase, alanineaminotransferase, aspartate aminotransferase and other NADH-based,FADH-based or oxidase-based assays), biological or chemical reductantsother than living cells which will reduce the reducible compound (forexample, ascorbates, cysteine, glutathione or thioredoxin),metabolizable substances (for example, glucose, lactic acid,triglycerides or cholesterol) and immunoreactants (for example,antigens, antibodies or haptens).

The compositions can be used to monitor enzyme redox reactions as wellas flavin adenine dinucleotide (FAD-FADH)-based and nicotinamide adeninedinucleotide (NAD-NADH)-based and (NADP-NADPH)-based reactions. In suchinstances, the reducible compound can be used to provide a dye in placeof NADH.

The reducible compounds of this invention, are particularly useful indetecting or quantifying living cells in biological samples. Althoughany biological sample suspected of having living cells therein can beanalyzed for bacteria, white blood cells, yeast or fungi by thisinvention, the invention is particularly useful for bacterial detectionin biological fluids, such as human and animal fluids (for example,urine, cerebral spinal fluid, blood as well as stool secretions) andsuspensions of human or animal tissue. The practice of this invention isparticularly important for detection of urinary tract infections inurine (diluted or undiluted).

When determining living cells using the reducible compounds, it ispreferable that the living cells interact with an electron transferagent (herein ETA). The presence of an ETA may also provide moreefficient dye release for analytical determinations of nonlivinganalytes. The ETA is a mobile compound which acts as an intermediarybetween the substance being determined (for example, living cell) andthe reducible compound.

In general, the potential of the ETA should be more positive than thepotential of the substance to be determined and less positive than thepotential of the reducible compound of this invention. That is, the ETAshould be more easily reduced than the analyte and less easily reducedthan the reducible compound. They are generally present at aconcentration that is dependant upon the concentration of the analyte,and preferably at a concentration of from about 1×10⁻³ molar to about1×10⁻⁷ molar.

ETA compounds useful in the practice of this invention include phenazinemethosulfate, phenazine ethosulfate and similar compounds known to oneskilled in the art. Combinations of different ETA compounds can be usedif desired. Preferred ETA compounds useful in the practice of thisinvention are the subject of U.S. Pat. No. 4,746,607 (issued May 24,1988 to Mura et al). In general, those compounds are substituted benzo-and naphthoquinones.

The detection of living cells, and particularly of bacterial cells, isoften carried out in the presence of a nutrient for those cells althoughits presence is not essential. Any nutrient media can be used whichcontains useful carbon, and optionally nitrogen, sources. Suitablenutrient media having proper components and pH are well known in theart. Particularly useful nutrients are glucose or tryptose alone or incombination.

The present invention is adaptable to either solution or dry assays. Ina solution assay, a solution (or dispersion) containing a reduciblecompound, and preferably an ETA, is prepared and contacted with a liquidtest sample containing the living cells or analyte to be determined bymixing. The ETA can also be mixed with the test sample prior to mixingwith the reducible compound. Generally the reducible compound is mixedwith the test sample in a suitable container (for example, test tube,petri disk beaker or cuvette). The resulting solution (or dispersion) isgently mixed and incubated for a relatively short time at a temperatureup to about 40° C. The test sample is then evaluated by measuring thedye at a suitable wavelength. Such an evaluation can be done withsuitable detection equipment.

A solution assay can also be carried out by contacting a porous,absorbent material, for example a paper strip, containing the testsample with a dispersion of the reducible compound. The analyte in thetest sample can migrate from the porous material into the dispersion andinitiate the analytical reactions needed for determination. In solutionassays, the amount of reducible compound present is at least about0.001, and preferably from about 0.01 to about 1.0, millimolar. Otherreagents can be present in amounts readily determined by one skilled inthe art.

Alternatively, the method of this invention can be practiced in a dryassay with a dry analytical element. Such an element can be an absorbentcarrier material, that is, a thin sheet or strip of self-supportingabsorbent or bibulous material, such as filter paper or strips, whichcontains the reducible compound or a dried residue of the dispersioncomprising same. Such elements are known in the art as test strips,diagnostic elements, dip sticks, diagnostic agents and the like.

When employed in dry analytical elements, the reducible compoundsdescribed herein can be incorporated into a suitable absorbent carriermaterial by imbibition or impregnation, or can be coated on a suitableabsorbent carrier material. Alternatively, they can be added to theelement during an assay. Useful carrier materials are insoluble andmaintain their structural integrity when exposed to water or biologicalfluids such as urine or serum. Useful carrier materials can be preparedfrom paper, porous particulate structures, cellulose, porous polymericfilms, wood, glass fiber, woven and nonwoven fabrics (synthetic andnonsynthetic) and the like. Useful materials and procedures for makingsuch elements are well known in the art as exemplified by U.S. Pat. Nos.3,092,465 (issued Jun. 4, 1963 to Adams et al), 3,802,842 (issued Apr.9, 1974 to Lange et al), 3,915,647 (issued Oct. 28, 1975 to Wright),3,917,453 (issued Nov. 4, 1975 to Milligan et al), 3,936,357 (issuedFeb. 3, 1976 to Milligan et al), 4,248,829 (issued Feb. 3, 1981 toKitajima et al), 4,255,384 (issued Mar. 10, 1981 Kitajima et al), and4,270,920 (issued Jun. 2, 1981 to Kondo et al), and U.K. Patent2,052,057 (published Jan. 21, 1981).

A dry assay can be practiced to particular advantage with an analyticalelement comprising a nonporous support having thereon at least oneporous spreading zone as the absorbent carrier material. The reduciblecompound can be in the spreading zone or in a different zone (forexample, reagent zone, registration zone or hydrophilic zone). Thespreading zone can be prepared from any suitable fibrous or non-fibrousmaterial or mixtures of either or both. The void volume and average poresize of this zone can be varied depending upon the use intended. Forexample, if whole blood or other liquid samples containing cells or highmolecular weight materials are to be assayed, the void volume andaverage pore size are generally greater than if serum or urine is to beassayed.

The spreading zone can be prepared using fibrous materials, either mixedwith a suitable binder material or woven into a fabric, as described inU.S. Pat. No. 4,292,272 (issued Sep. 29, 1981 to Kitajima et al), frompolymeric compositions (for example, blush polymers) or particulatematerials, with or without binding adhesives, as described in U.S. Pat.Nos. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al), 4,258,001(issued Mar. 24, 1981 to Pierce et al) and 4,430,436 (issued Feb. 7,1984 to Koyama et al) and Japanese Patent Publication 57(1982)-101760(published Jun. 24, 1982). It is desired that the spreading zones beisotropically porous, meaning that the porosity is the same in eachdirection in the zone as created by interconnected spaces or poresbetween particles, fibers or polymeric strands.

The dry analytical element of this invention can be a singleself-supporting porous spreading zone containing a reducible compoundand any other desired reagents for a particular use, but preferably suchzone is carried on a suitable nonporous support. Such a support can beany suitable dimensionally stable, and preferably, transparent (that is,radiation transmissive) film or sheet material which transmitselectromagnetic radiation of a wavelength between about 200 and about900 nm. Useful support materials include polystyrene, polyesters,polycarbonates and cellulose esters.

The elements can have more than one zone which are generally in fluidcontact with each other, meaning that fluids, reagents and reactionproducts can pass between superposed regions of adjacent zones.Preferably, the zones are separately coated superposed layers, althoughtwo or more zones can be in a single coated layer. Besides the patentsnoted above, suitable element formats and components are described also,for example, in U.S. Pat. Nos. 4,042,335 (issued Aug. 16, 1977 toClement) and 4,144,306 (noted above) and Re. No. 30,267 (reissued May 6,1980 to Bruschi).

In the elements of this invention, the amount of the reducible compoundcan be varied widely, but it is generally present in a coverage of atleast about 0.01, and preferably from about 0.05 to about 0.2, g/m².Optional, but preferred reagents are generally present in the followingcoverages:

ETA: generally at least about 0.001, and preferably from about 0.01 toabout 1, g/m²,

nutrient: generally at least about 0.05, and preferably from about 0.1to about 2, g/m² (used only in living cell detection),

buffer (pH≦9): generally at least about 0.1, and preferably from about0.5 to about 2, g/m², and

surfactant: generally at least about 0.1, and preferably from about 0.2to about 5, g/m².

One or more of the zones can contain a variety of other desirable, butoptional, components, including activators, binders (generallyhydrophilic), antioxidants, coupler solvents, etc. as is known in theart, as well as any reagents needed for assay of a particular analyte.

In one embodiment of this invention, an element for detection ofmicroorganisms in an aqueous liquid comprises an electron transfer agentand a reducible compound, both of which are described above. It isdesirable that these elements also contain a nutrient for the livingcells and a buffer which maintains physiological pH during the assay.Such an element can be used to detect bacteria, for example, in a urinesample (for example, one pretreated to eliminate reductive interferents)by physically contacting the sample and element in a suitable manner,and detecting the detectable species released from the reduciblecompound as a result of the presence of the bacteria at the appropriatewavelength.

In another embodiment of this invention, an element is used for thedetermination of a nonliving biological or chemical analyte in anaqueous liquid. An interactive composition containing one or morereagents can be incorporated into the element or added at the time ofthe assay. This composition contains the reagents needed to interactwith the analyte to bring about reduction of the reducible compound andeventual dye formation. Examples of such analytes are described above.The amount of dye detected can be correlated to the amount of analytepresent in the liquid sample.

The element of this invention is also useful for determining otherreductants such as ascorbate (ascorbic acid and equivalent alkali metalsalts), cysteine, glutathione, thioredoxin and the like.

A variety of different elements, depending on the method of assay, canbe prepared in accordance with the present invention. Elements can beconfigured in a variety of forms, including elongated tapes of anydesired width, sheets, slides or chips.

The assay of this invention can be manual or automated. In general, inusing the dry elements, an analyte or living cell determination is madeby taking the element from a supply roll, chip packet or other sourceand contacting it with a sample (for example, 1-200 μl) of the liquid tobe tested so that the sample mixes with the reagents in the element.Such contact can be accomplished in any suitable manner, for example, bydipping or immersing the element into the sample or, preferably, byspotting the element by hand or machine with one or more drops of thesample with a suitable dispensing means.

After sample application, the element is exposed to any conditioning,such as incubation, heating or the like, that may be desirable toquicken or otherwise facilitate obtaining any test result.

Detection of an analyte or living cell is achieved when the reduciblecompound is reduced releasing a leuco dye which in turn is oxidized byanother molecule of reducible compound to provide a dye which can bedetected in a suitable manner. Spectral determinations can be madeeither at the maximum wavelength of the dye or at wavelengths other thanthe maximum wavelength.

Reagents used in the following examples were obtained as follows:ascorbic acid, sodium salt, nicotinamide adenine dinucleotide, reducedform (NADH) and phenazine methosulfate from Sigma Chemical Co., brainheart infusion (BHI) media from Difco Labs, TRITON X-100 surfactant fromRohm & Haas, titanium (IV) chloride and sodium borohydride (10% onalumina) from Morton-Thiokol, Inc., Alfa Products, and the bacterialmicroorganisms from the American Type Culture Collection (ATCC) inRockville, Md. All other reagents were either obtained from EastmanKodak or prepared using known starting materials and procedures.

Escherichia coli (ATCC 25922) cells were grown in BHI medium at 37° C.without shaking and transferred daily. Forty ml of the cells grownovernight were harvested by centrifugation and resuspended in 10 ml of a0.05 molar potassium phosphate buffer (pH 7.5). Solution 1 was preparedby adding 5 ml of the cell suspension to 9 ml of buffer. Solution 2 wasprepared by adding 1 ml of solution 1 to 9 ml of buffer. Solutions 2a,2b, 2c, and others, were prepared by a 1:1 dilution of Solution 2. Theturbidity of each solution was measured at 620 nm against a buffer blankin a commercially available Beckman A25 spectrophotometer. A linearrelationship between turbidity measurement and viable cell counts hadbeen predetermined. An absorbance of 0.1 was found to be equivalent toabout 6×10⁷ E. coli cells/ml. Using this relationship and the knowndilution factor, the number of cells in Solution 1 was determined.

The following examples are presented to illustrate the practice of thisinvention.

EXAMPLE 1 Preparation of Compound I

Compound I identified above was prepared as follows:

Preparation of Leuco Methylene Blue Carbamyl Chloride Intermediate:

Methylene blue (5.5 g, 0.017 mole) was mixed with 2,6-lutidine (5 ml)and palladium catalyst (200 mg, on carbon) in dry acetonitrile (200 ml)in an hydrogenation bottle under hydrogen pressure. This mixture washydrogenated with vigorous shaking at 3 atmospheres hydrogen pressureuntil a colorless solution was obtained. The resulting solution wasrapidly filtered into a stirred solution of phosgene (20 ml of a 1 molarsolution in toluene) and dry acetonitrile (50 ml) while maintaining anitrogen atmosphere over the solution. A white solid precipitated, wascollected, washed with acetonitrile and dried to give 2.6 g ofintermediate.

Preparation of Hydroxymethyl Quinone Intermediate:

The following reaction sequence was followed to prepare thisintermediate. ##STR5##

Compound A (identified above) was prepared by the procedure described incopending and commonly assigned U.S. Ser. No. 824,766 (filed Jan. 31,1986 by Belly et al), Example 21 and Example 1 (Steps 2a, 2b and 3).

Compound A (25.2 g, 86.4 mmole), iodomethane (49 g, 346 mmole) andpotassium carbonate (35.8 g, 259 mmole) were heated at reflux in acetone(150 ml) for 24 hours. The reaction mixture was allowed to cool and thenpoured into dilute hydrochloric acid in ice/water (200 ml). Theprecipitated solid was collected by filtration and washed with water,air dried and recrystallized from ethanol with decolorizing carbon togive 16.75 g (60.6% yield) of Compound B (m.p. 186°-187° C.). Thestructure (identified above) was confirmed by NMR.

Titanium (IV) chloride (38 g, 0.2 mole) was added to an ice coldsolution of Compound B (32 g, 0.1 mole) in dichloromethane (750 ml),stirring under a nitrogen atmosphere. To this solution was addeddropwise α,α-dichloromethylmethylether (18.9 g, 0.165 mole), and thereaction mixture was allowed to warm to room temperature over about 15hours. The mixture was again cooled and ice water (1 liter) wascarefully added dropwise. The resulting layers were separated and thewater layer was washed with dichloromethane. The combined organic layerswere dried and the solvent was removed. The crude product was purifiedby chromatography (silica; dichloromethane) and the resulting solid wastriturated in cold diethylether, filtered and washed with colddiethylether to give Compound C (16.1 g, 46.5% yield) as a while solid(m.p. 208°-210° C.). The structure (identified above) was confirmed byNMR.

Sodium borohydride (10% on alumina, 27.9 g, 73.5 mmole) was added to asuspension of Compound C (15 g, 43.2 mmole) in ethyl acetate (400 ml).After stirring for one hour at room temperature, the reaction mixturewas filtered and the alumina was washed with ethylacetate. The filtratewas freed of solvent to give 16 g of crude Compound D. The structure(identified above) was confirmed by NMR.

Ceric ammonium nitrate (75.3 g, 137 mmole) in water (150 ml) was rapidlyadded dropwise to a suspension of Compound D (16 g, 45.8 mmole) inacetonitrile (265 ml) and water (15 ml). After stirring two hours atroom temperature, the reaction mixture was poured into ice/water (500ml) and the precipitated product was collected, washed with water andair dried. The crude product was purified by chromatography (silica;dichloromethane/diethylether), and filtered to give 9.4 g (64% yield) ofCompound E (m.p. 219°-221° C.). The structure (identified above) wasconfirmed by NMR.

Preparation of Compound I:

Hydroxymethylquinone (2.23 g, 7 mmole), leuco methylene blue carbamylchloride (2.44 g, 7 mmole) and 4-(N,N-dimethylamino)pyridine (0.85 g, 7mmole) were dissolved in dichloromethane (50 ml) and cooled to 0° C.1,8-diazabicycloundec-7-ene (1.06 g, 7 mmole) was added, and theresulting mixture was stirred at 0° C. for 25 minutes. A small amount ofacetic acid in water was added to neutralize the bases before dilutingthe mixture with ice water and dichloromethane. The organic phase waswashed several times with cold water to remove methylene blue beforedrying over magnesium sulfate. The crude product was chromatographed onsilica gel using 1% diethylether in dichloromethane as eluent. One gramof glassy product was obtained. Half of this product was slurried indiethylether/ethanol with two equivalents of concentrated hydrochloricacid to precipitate a gummy solid salt. This salt was slurried with atwo-phase mixture of diethylether and water. The diethylether phase wasremoved and stripped to a solid (100 mg) Compound I. Thin layerchromatography demonstrated this solid to be reasonably pure. Theresulting reducible compound having a leuco dye moiety was reasonablystable as a solid, but decomposed upon standing in a solution. NMRsubstantiated the structure. When contacted with ascorbic acid, anintense blue color was observed when the compound was warmed.

EXAMPLE 2 Preparation of Compound II

Compound II identified above was prepared using the following procedure:

Preparation of Intermediate Compound:

A sample of the yellow dye (2.7 g, 0.01 mole) ##STR6## 2,6-lutidine(2.14 g, 0.02 mole), 10% platinum on carbon (300 mg), a small stir bar,tetrahydrofuran (30 ml) and dichloromethane (70 ml) were added to a Parrbottle and subjected to 3 atmospheres of hydrogen pressure for about oneminute until the color of the solution had faded from red to a paleyellow. The bottle was removed from the shaker under a nitrogenatmosphere and cooled to -78° C. with dry ice/isopropanol. A solution(10 ml) of a 1 molar solution of COCl₂ in toluene was added beforewarming the mixture to 0° C. for 20 minutes. The catalyst as well assome undissolved dye were removed by filtration before washing thesolution with cold aqueous hydrochloric acid. After drying overmagnesium sulfate, the solution was concentrated in vacuo, and dilutedwith a little diethylether to produce 1.2 g of solid carbamyl chloride.Thin layer chromatography showed the solid to contain some startingyellow dye. The solid was used directly in the next step.

Preparation of Compound II:

Crude intermediate carbamyl chloride (1.2 g), hydroxymethylquinone (638mg, 2 mmole), 4-(N,N-dimethylamino)pyridine (244 mg, 2 mmole) and1,8-diazabicycloundec-7-ene (304 mg, 2 mmole) were reacted in 10 ml ofdichloromethane as in Example 1. The procedure of Example 1 was followedto provide crude product which was chromatographed on silica using 4%diethylether in dichloromethane. After slurrying indiethylether/methanol and then boiling acetonitrile, the eluted materialwas converted into 480 mg of yellow powder. NMR confirmed the structureof Compound II which was more stable than Compound I.

EXAMPLE 3 Detection of Microorganism in Solution

This is a comparative example demonstrating the practice of the presentinvention to detect E. coli in an solution assay compared to a similarassay carried out according to U.S. Ser. No. 824,766, noted above.

The reducible compound of the noted application used herein in theControl assay was ##STR7## E. coli cells were cultured as describedabove. A dispersion of the Control reducible compound was prepared bydissolving the compound (16 mg) in N,N-dimethylformamide (1 ml) whichhad been acidified with 0.1% sulfuric acid. A sample (100 μl) of thisdispersion was added to TRITON X-100 nonionic surfactant (200 μl), andthis second solution was added to potassium phosphate buffer (9.7 ml, pH7.5) with stirring. A dispersion of the reducible compound II of thisinvention was similarly prepared except that 10.25 g of the compound wasused.

Test solutions were prepared from the following: dispersion as describedabove (1.5 ml), glucose solution (25 μl, 10% in water),trimethyl-1,4-benzoquinone ETA (0.01 molar in methanol, 25 μl),potassium phosphate buffer (1.15 ml, 0.05 molar, pH 7.5) and E. colicell suspension (final concentration of 5×10⁵ cells/ml). Backgroundsolutions contained all reagents except microbial cells.

Optical densities were determined at 37° C. in a standardspectrophotometer at 650 nm for the Control assay and at 450 nm for theassay of this invention. The results are shown in Table I below as thechange in optical density (ΔOD) after thirty minutes, corrected forbackground. The assay of this invention exhibited almost a 200% increasein cell response over the Control assay. This indicates a significantlygreater sensitivity for the assay of this invention over the prior artassay.

                  TABLE I                                                         ______________________________________                                                      ΔOD After 30                                              Assay         Minutes, 37° C.                                          ______________________________________                                        Control       0.039                                                           Example 3     0.114                                                           ______________________________________                                    

EXAMPLES 4 and 5 Assays for Ascorbic Acid and NADH

These are comparative examples demonstrating the practice of thisinvention for the detection of ascorbic acid (Example 4) and NADH(Example 5) as compared to a Control assay using a reducible compound asdescribed in Example 3 above.

A dispersion of the Control reducible compound was prepared bydissolving it in N,N-dimethylformamide (16 mg/ml), followed by addingthe resulting solution (100 μl) to TRITON X-100 nonionic surfactant (200μl). This solution was then added to a solution containing sodiumphosphate buffer (9.3 ml, pH 7.5), 10% glucose solution (200 μl) andtrimethyl-1,4-benzoquinone or phenazine methosulfate ETA (0.01 molar).The phenazine methosulfate was used for detection of NADH while thebenzoquinone was used for ascorbate detection. A dispersion of thereducible compound of this invention was similarly prepared except that10.25 mg/ml was used.

Test solutions were prepared as follows: dispersion (1.5 ml) potassiumphosphate buffer (1.4 ml, pH 7.5), and either ascorbate or NADH (100μl). A background solution contained all reagents except the analyte(ascorbate or NADH).

Optical densities were determined at 37° C. using a standardspectrophotometer at 635 nm for the Control assay and at 500 nm for theassay of this invention. The results are shown in Tables II and III asthe change in optical density (ΔOD) after 30 minutes and corrected forbackground. A substantial increase in response was seen for both assaysof this invention over the Control assay.

                  TABLE II                                                        ______________________________________                                                   Analyte                                                                       Concentration                                                                             ΔOD After 30 Minutes,                            Assay      (molar)     37° C.                                          ______________________________________                                        Control    3.3 × 10.sup.-6                                                                     0.038                                                  Example 4  3.3 × 10.sup.-6                                                                     0.088                                                  Control    3.3 × 10.sup.-7                                                                     0.002                                                  Example 4  3.3 × 10.sup.-7                                                                     0.018                                                  ______________________________________                                    

                  TABLE III                                                       ______________________________________                                                   Analyte                                                                       Concentration                                                                             ΔOD After 30 Minutes,                            Assay      (molar)     37° C.                                          ______________________________________                                        Control    3.3 × 10.sup.-6                                                                     0.146                                                  Example 5  3.3 × 10.sup.-6                                                                     0.702                                                  Control    3.3 × 10.sup.-7                                                                     0.015                                                  Example 5  3.3 × 10.sup.-7                                                                     0.082                                                  ______________________________________                                    

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be effected within the spirit and scope of theinvention.

I claim:
 1. A composition for chemical analysis buffered at a pH of 9 orless and comprising an electron transfer agent, and a reducible compoundof the structure CAR--R¹ wherein CAR-- is the quinone structure ##STR8##R² and R⁴ are independently hydrogen, alkyl of 1 to 40 carbon atoms,aryl of 6 to 10 carbon atoms in the aromatic ring or an electronwithdrawing group having a Hammett sigma value greater than about 0.06and selected from the group consisting of cyano, carboxy, nitro, halo,trihalomethyl, trialkylammonium, carbonyl, carbamoyl, sulfonyl, alkyl of1 to 40 carbon atoms substituted with at least one of said foregoinggroups, and aryl having 6 to 10 carbon atoms in the aromatic ringsubstituted with at least one of said foregoing groups,R⁵ is methylene,R³ is the same as --R⁵ --R¹, or is hydrogen, alkyl of 1 to 40 carbonatoms, aryl of 6 to 10 carbon atoms in the aromatic ring or an electronwithdrawing group having a Hammett sigma value greater than about 0.06and selected from the group consisting of cyano, carboxy, nitro, halo,trihalomethyl, trialkylammonium, carbonyl, carbamoyl, sulfonyl, alkyl of1 to 40 carbon atoms substituted with at least one of said foregoinggroups, and aryl having 6 to 10 carbon atoms in the aromatic ringsubstituted with at least one of said foregoing groups, or R³ and R⁴,taken together, represent the atoms necessary to complete a fusedcarbocyclic ring attached to the quinone nucleus, said ring having 4 to8 carbon atoms in its backbone, R⁶ is aryl of 6 to 10 carbon atoms inthe aromatic ring, and R⁷ is substituted aryl, anilino, naphthylimino oran activated methylene group, or R⁶ and R⁷, taken together with thenitrogen atom to which they are attached, represent the carbon andhetero atoms which, after release of R¹ from said quinone nucleus anddecarboxylation, form a leuco dye.
 2. A dry analytical element for thedetermination of an analyte which is oxidizable or capable of producingan oxidizable species, said analyte selected from the group consistingof microorganisms, white blood cells, yeast, fungi, enzymes, ascorbates,cysteine, glutathione, thioredoxin, metabolites and immunoreactants,saidelement comprising an absorbent carrier material and one or morereaction zones, said element also containing in one of said zones, areducible compound of the structure CAR--R¹ wherein CAR-- is the quinonestructure ##STR9## R² and R⁴ are independently hydrogen, alkyl of 1 to40 carbon atoms, aryl of 6 to 10 carbon atoms in the aromatic ring or anelectron withdrawing group having a Hammett sigma value greater thanabout 0.06 and selected from the group consisting of cyano, carboxy,nitro, halo, trihalomethyl, trialkylammonium, carbonyl, carbamoyl,sulfonyl, alkyl of 1 to 40 carbon atoms substituted with at least one ofsaid foregoing groups, and aryl having 6 to 10 carbon atoms in thearomatic ring substituted with at least one of said foregoing groups, R⁵is methylene, R³ is the same as --R⁵ --R¹, or is hydrogen, alkyl of 1 to40 carbon atoms, aryl of 6 to 10 carbon atoms in the aromatic ring or anelectron withdrawing group having a Hammett sigma value greater thanabout 0.06 and selected from the group consisting of cyano, carboxy,nitro, halo, trihalomethyl, trialkylammonium, carbonyl, carbamoyl,sulfonyl, alkyl of 1 to 40 carbon atoms substituted with at least one ofsaid foregoing groups, and aryl having 6 to 10 carbon atoms in thearomatic ring substituted with at least one of said foregoing groups, orR³ and R⁴, taken together, represent the atoms necessary to complete afused carbocyclic ring attached to the quinone nucleus, said ring having4 to 8 carbon atoms in its backbone, R⁶ is aryl of 6 to 10 carbon atomsin the aromatic ring, and R⁷ is substituted aryl, anilino, naphthyliminoor an activated methylene group, or R⁶ and R⁷, taken together with thenitrogen atom, represent the carbon and hetero atoms which, afterrelease of R¹ from said quinone nucleus and decarboxylation, form aleuco dye.
 3. The element of claim 2 further comprising an electrontransfer agent.
 4. The element of claim 2 further comprising an nutrientmetabolizable by microorganisms.
 5. The element of claim 2 having areducible compound wherein at least two of R², R³ and R⁴ areindependently electron withdrawing groups, or R³ and R⁴, taken together,represent the atoms necessary to complete a fused 5- to 7-membercarbocyclic ring attached to the quinone nucleus.
 6. The element ofclaim 5 having a reducible compound wherein R³ and R⁴, taken together,represent the atoms necessary to complete a fused 5- to 7-memberedcarbocyclic ring attached to the quinone nucleus.
 7. The element ofclaim 2 wherein the reducible compound is selected from the groupconsisting of: ##STR10##
 8. A method for the determination of an analytewhich is oxidizable or capable of producing an oxidizable species, saidmethod comprising the steps of:A. at a pH of 9 or less, contacting asample of a liquid suspected of containing an analyte which isoxidizable or capable of producing an oxidizable species, said analyteselected from the group consisting of microorganisms, white blood cells,yeast, fungi, enzymes, ascorbates, cysteine, glutathione, thioredoxin,metabolites and immunoreactants,with a reducible compound of thestructure CAR--R¹ wherein CAR-- is the quinone structure ##STR11## R²and R⁴ are independently hydrogen, alkyl of 1 to 40 carbon atoms, arylof 6 to 10 carbon atoms in the aromatic ring or an electron withdrawinggroup having a Hammett sigma value greater than about 0.06 and selectedfrom the group consisting of cyano, carboxy, nitro, halo, trihalomethyl,trialkylammonium, carbonyl, carbamoyl, sulfonyl, alkyl of 1 to 40 carbonatoms substituted with at least one of said foregoing groups, and arylhaving 6 to 10 carbon atoms in the aromatic ring substituted with atleast one of said foregoing groups, R⁵ is methylene, R³ is the same as--R⁵ --R¹, or is hydrogen, alkyl of 1 to 40 carbon atoms, aryl of 6 to10 carbon atoms in the aromatic ring or an electron withdrawing grouphaving a Hammett sigma value greater than about 0.06 and selected fromthe group consisting of cyano, carboxy, nitro, halo, trihalomethyl,trialkylammonium, carbonyl, carbamoyl, sulfonyl, alkyl of 1 to 40 carbonatoms substituted with at least one of said foregoing groups, and arylhaving 6 to 10 carbon atoms in the aromatic ring substituted with atleast one of said foregoing groups, or R³ and R⁴, taken together,represent the atoms necessary to complete a fused carbocyclic ringattached to the quinone nucleus, said ring having 4 to 8 carbon atoms inits backbone, R⁶ is aryl of 6 to 10 carbon atoms in the aromatic ring,and R⁷ is substituted aryl, anilino, naphthylimino or an activatedmethylene group, or R⁶ and R⁷, taken together with the nitrogen atom towhich they are attached, represent the carbon and hetero atoms which,after release of R¹ from said quinone nucleus and decarboxylation, forma leuco dye, B. oxidizing said leuco dye with additional molecules ofsaid reducible compound to provide a dye, and C. detecting the provideddye as a result of the presence of said analyte which effects therelease of R¹ from said reducible compound.
 9. The method of claim 8using a reducible compound wherein at least two of R², R³ and R⁴ areindependently electron withdrawing groups, or R³ and R⁴, taken together,represent the atoms necessary to complete a fused 5- to 7-membercarbocyclic ring attached to the quinone nucleus.
 10. The method ofclaim 9 using a reducible compound wherein R³ and R⁴, taken together,represent the atoms necessary to complete a fused 5- to 7-memberedcarbocyclic ring attached to the quinone nucleus.
 11. The method ofclaim 8 wherein the reducible compound is selected from the groupconsisting of: ##STR12##
 12. The method of claim 8 for the determinationof microorganisms in the presence of an electron transfer agent.
 13. Themethod of claim 8 for the determination of microorganisms in thepresence of a metabolizable nutrient.
 14. The method of claim 13 for thedetermination of microorganisms in a urine sample.